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| Status |
Public on Jun 29, 2011 |
| Title |
Neocortex_1 |
| Sample type |
SRA |
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| Source name |
Neocortex
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| Organism |
Mus musculus |
| Characteristics |
background strain: C57BL/6 & BALB/c
mouse age: P56
tissue: Neocortex
number of pooled tissues: 1
ip antibody: anti-Ago2 (2E12-1C9, Abnova)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Neocortex and cerebellum of P56 mouse brain were dissected on ice and flash frozen in liquid nitrogen, ground to a fine powder, and resuspend in 10 volume of ice-cold lysis buffer (10mM HEPES pH7.4, 100mM KCl, 5mM MaCl2, 0.5%NP-40, 1mM DTT, 100U/ml RNasin) containing Roche Complete proteinase inhibitors, EDTA-free. Tissue suspension was homogenized using glass douncer. Homogenates were centrifuged for 30min at 13,000g, 4C to pellet cell debris and unsolubilized material. Mouse-anti-c-Myc(sc-40, Santa Cruz Biotechnology), mouse-anti-Ago2(2E12-1C9, Abnova) or mouse IgG1(Molipore) conjugated protein G Dynabeads(Invitrogen) were added into supernatant, and the mixture was incubated in 4C with end-over-end rotation for 4 hours. Beads were washed twice with low salt NT2 buffer(50mM Tris.Hcl pH 7.5, 150mM NaCl, 1mM MgCl2, 0.5%NP-40, 1mM DTT, 100U/ml RNasin) and twice with high salt NT2 buffer(50mM Tris.Hcl pH 7.5, 600mM NaCl, 1mM MgCl2, 0.5%NP-40, 1mM DTT, 100U/ml RNasin) and treated with 0.6mg/ml proteinase K for 20min at 55C. RNA was extracted by acid phenochloroform(Ambion), followed by chloroform, and precipitated with sodium acetate and glycoblue(Ambion) in ethanol overnight -80C. RNA pellet was washed once in 75% ethanol and resuspend in water for further application.RNA was successively ligated to 3â and 5â adaptors, gel purified after each ligation, reverse transcribed and PCR amplified using Solexa sequencing primers. PCR product was gel purified, quantified , and sequenced for 36 cycles on Illumina Genome Analyzer II. Radiolabeled 19nt and 21nt RNA oligos were used to trace RNA for gel purification, and were depleted by PmelI digestion after PCR amplification.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
19-24nt
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| Data processing |
Raw Illumina sequencing reads were trimmed from 3' linker, filtered for low-quality reads, and collapsed to unique sequences retaining their individual read count information. Unique sequences 18nt or longer in length were mapped to the University of California at Santa Cruz mm9 assembly of the mouse genome using bowtie allowing no mismatch. miRNA annotations were made according to miRbase version 16.[miRNA_annotation.txt is provided as supplementary file on Series records]
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| Submission date |
Jun 28, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Miao He |
| E-mail(s) |
hem@cshl.edu
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| Organization name |
Cold Spring Harbor Laboratory
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| Department |
Neuroscience
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| Lab |
Z. Josh huang
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| Street address |
1 bungtown road
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| City |
cold spring harbor |
| State/province |
new york |
| ZIP/Postal code |
11724 |
| Country |
USA |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE30286 |
Cell-type based analysis of microRNA profiles in the mouse brain |
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| Relations |
| SRA |
SRX081799 |
| BioSample |
SAMN00632050 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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